Diagnostic, research and therapeutic applications often require the purification or separation of one or more proteins from more complex mixtures, for example, mixtures derived from biological tissues, biological fluids, cell cultures and the like. Various techniques which typically exploit differences in physical properties as between proteins in the mixture, such as charge, hydrophobicity, size, shape, or affinity for a particular ligand have been proposed for protein separation.
Column chromatography is one method commonly used to separate protein mixtures. In this method, a sample containing a mixture of proteins is typically passed through a stationary bed of chromatographic resin; in general, proteins having relatively less affinity for the resin elute first, followed by proteins having relatively greater affinity for the resin. Examples include reversed-phase high-performance liquid chromatography and anion exchange column chromatography. For example, in ion exchange chromatography, positively charged proteins tend to be retained by a cation exchange column whereas proteins having a negative or neutral charge pass through the column, and negatively charged proteins tend to be retained by an anion exchange column whereas proteins having a positive or neutral charge pass through the column. Flow rates through ion exchange columns, however, tend to be relatively slow. One of the restrictions of separating proteins using anion exchange column chromatography is the inability of using high concentrations of urea in the extraction reagent or in the buffers. This high concentration of urea is required to solubilize the proteins that have a low aqueous solubility but when injected in a column chromatography will generate a high backpressure. This prefractionation is therefore limited to the soluble proteins.
An alternative to column chromatography is batch chromatography. In batch chromatography, a quantity of ion exchange resin is added directly to a sample to form a dispersion of the resin in the sample and the dispersion is then agitated or gently mixed. The protein-bound resin is removed from the remainder of the sample by centrifugation or filtration, leaving unbound protein in solution. See, for example, Chang, U.S. Pat. No. 5,250,662, and Larsen, et al., U.S. Pat. No. 5,888,966. Again, however, times required for the separation may be undesirably long.
Another common technique used for protein separation is electrophoresis. In electrophoresis, proteins migrate in an electric field at rates that depend on their net charge, size, and/or shape. There are several different techniques that fall under the heading of electrophoresis that are well known in the art, including gel electrophoresis (PAGE) and SDS-electrophoresis (SDS-PAGE). One such technique uses a multi-compartment electrolyzer (MCE) and isoelectric membranes to separate proteins based on charge. See Herbert, B., Righetti, P. G., Electrophoresis, 21, pp. 5641-5649 (2002). This isoelectric membrane technique, in combination with other separation techniques, allows for visualization of proteins, as a result of enhanced resolution, and to some extent, for the visualization of less abundant proteins, as a result of greater detection sensitivity. The isoelectric membrane technique, however, has significant disadvantages; it is relatively complex, requiring an electrode potential and specialized, expensive instrumentation, and is time-consuming, requiring up to twenty-four hours for completion.
Two-dimensional gel electrophoresis (2-DE) is an analytical technique that simultaneously separates thousands of proteins and allows comparative protein profiling between different crude biological samples. The existing 2-DE gel methods are incapable of detecting the majority of protein components in complex proteomes such as mammalian cells, tissues, and biological fluids. To increase the resolution of 2-DE separation, long Immobilized pH gradient (IPG) strips with narrow pH ranges have been designed. After separating the proteins by 2-DE, the resultant spots can be cut, destained, digested by trypsin, and analyzed by Matrix-Assisted Laser Desorption Ionization-Time of Flight-Mass Spectrometry (MALDI-TOF-MS). Tools like ExPASy, that scan the different protein databases available, allow for the identification of the protein based on its isoelectric point (pI), molecular mass (Mr), and the masses of the different peptides generated from the trypsin digestion of this protein. The pI and Mr can be estimated from the location of the spot on the gel. The masses of the peptide fragments are measured by MALDI-TOF-MS.
One of the main limitations that might hinder the identification of a protein separated by 2-DE is its low abundance in the sample. This limitation is a result of the finite protein loading limits on commercially available Immobilized pH Gradient (IPG) strips, where the first dimension of the separation occurs. The maximum protein load on an 11 cm IPG strip is less than 300 μg, but when an IPG strip (pH 7 to 10) is used, many proteins contained in the loading capacity, particularly those with pIs lower than 7 and higher than 10, will not be separated. Proteins outside the boundaries of the pH gradient will be electrostatically pushed to either end of the IPG strip because they remain net positively- or negatively-charged at every point of the IPG polyacrylamide gel. Since the proteins with pI values between 7 and 10 are a minority in the sample, many will be dragged by the overwhelming majority of proteins migrating to the ends of the strip. At the end of the separation, the amount of proteins profiled represents a fraction well below the original mass that was loaded.